Apparatus and method for detecting, quantifying and characterizing microorganisms

ABSTRACT

A method and apparatus for performing microbial antibiotic susceptibility testing include disposable, multi-chambered susceptibility plates and an automated plate handler and image acquisition and processing instrument is described. The susceptibility plates are inoculated with a suitable microorganism such as bacteria, fungi, protozoa, algae or viruses and anti-microbial agent(s) are applied that the microorganism is exposed to at a variety of concentrations or a gradient of each anti-microbial agent. The plates are then placed in the instrument, which monitors and measures the growth or lack thereof of the microorganisms. This data is used to determine the susceptibility of the microorganism to the antibiotics. Such a system automates anti-microbial susceptibility testing using solid media and Carbo-Bauer standardized result reporting. The system provides a level of automation previously associated only with broth micro dilution testing, while retaining the advantages of the manual disk diffusion test.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/364,903, filed Jul. 30, 1999 (now abandoned), which is acontinuation-in-part of U.S. application Ser. No. 09/267,863, filed Mar.12, 1999 (now U.S. Pat. No. 6,153,400).

This application is related to U.S. patent application Ser. No.08/989,560 to Jeffrey et al. filed Dec. 12, 1997, and U.S. patentapplication Ser. No. 09/267,863 to Matsumura et al., filed Mar. 12,1999, the subject matter of each being incorporated herein by reference.

BACKGROUND OF THE INVENTION

The presence of microbial contamination in clinical specimens isconventionally determined by culturing the specimens in the presence ofnutrients and detecting microbial activity through changes in thespecimen or in the atmosphere over the specimen after a period of time.For example, in the U.S. Pat. No. 4,182,656 to Ahnell et al., the sampleis placed in a container with a culture medium comprising a carbon 13labeled fermentable substrate. After sealing the container andsubjecting the specimen to conditions conducive to biological activity,the ratio of carbon 13 to carbon 12 in the gaseous atmosphere over thespecimen is determined and compared with the initial ratio. In U.S. Pat.No. 4,152,213, a method is claimed by which the presence of oxygenconsuming bacteria in a specimen is determined in a sealed container bydetecting a reduction in the amount of oxygen in the atmosphere over thespecimen through monitoring the pressure of gas in the container. U.S.Pat. No. 4,073,691 provides a method for determining the presence ofbiologically active agents, including bacteria, in a sealed containercontaining a culture medium by measuring changes in the character if thegaseous atmosphere over the specimen after a period of time.

A method for non-invasive detection is taught by Calandra et al., U.S.Pat. No. 5,094,955, where a device is disclosed for detecting thepresence of microorganisms in clinical specimens, such as blood or otherbody fluids, and in non-clinical specimens, by culturing the specimenswith a sterile liquid growth medium in a transparent sealed container.The presence of microorganisms is determined by detecting or measuringchanges in the pH of the specimen or the production of carbon dioxidewithin the specimen using a sensor affixed to the interior surface ofthe container or to the sealing means used to seal the container. InCalandra et al., microorganisms can be detected in the presence ofinterfering material, such as large concentrations of red blood cells,through non-radiometric and non-invasive means.

One disadvantage of the detection system of Calandra et al., is that thetime required for detecting the presence of microorganisms is related tothe number of microorganisms within the sample. Also, because the growthmedium for the microorganisms is a liquid, the container must usually beagitated during incubation, which is an additional expense involved inmaking the incubation equipment, as well as an increase in thelikelihood of a mechanical breakdown. Also, such a system allows for thedetermination of the presence of microorganisms, but does not allow forenumeration. Furthermore, it is often the case that after detection ofmicroorganisms, it is desired to identify the microorganisms and/ordetermine their susceptibility to various antibiotics. In aCalandra-type system, it would be necessary to plate out themicroorganisms from the liquid culture medium before performingsusceptibility or identification tests, which involves additionaltime—time that is not always available if the patient is very ill. Also,a Calandra-type system could not serve the additional functions ofreading/imaging plates for antibiotic susceptibility and/or microbialidentification.

Following detection of a microorganism in a patient sample, it is oftendesirable to determine to which antibiotics the microorganism issusceptible. There are now a number of bacterial species whichincreasingly exhibit resistance to one or more classes of antimicrobialagents, making it that much more important to perform susceptibilitytesting. Failure of a particular susceptibility test to accuratelypredict antimicrobial resistance in a patient's isolate couldsignificantly impact patient care if an antibiotic is used to which themicroorganism is not susceptible.

Different types of susceptibility tests can be used to test amicroorganism. The following brief descriptions give details of someknown susceptibility tests as well as some details that relate to thepresent invention.

One type of susceptibility test is the disk diffusion test, oftenreferred to as the Kirby-Bauer test. This is a standardized test thatinvolves inoculating (with 0.5 McFarland standardized suspension of amicrobial isolate) a gel plate (e.g. a 150-mm Mueller-Hinton agar plate)and placing thereon one or more disks impregnated with fixedconcentrations of antibiotics. After incubation (e.g. 18-24 hours at 35degrees C.), the diameter of zones of inhibition around the disks (ifpresent) determine the sensitivity of the inoculated microorganism tothe particular antimicrobial agent impregnated in each disk. Due to thestandardization of the Kirby-Bauer method, results of this method areanalyzed by comparing the diameter of the inhibition zone withinformation published by NCCLS (National Committee on ClinicalLaboratory Standards) in Performance Standards for Antimicrobial DiskSusceptibility Testing, the subject matter of which is incorporatedherein by reference. The results of this test are semi-quantitative inthat there are three categories of susceptibility—namely resistant,intermediate and susceptible. As can be seen in FIG. 14, an agar plate110 with inoculum has a plurality of disks 112 placed thereon, whichdisks are impregnated with antibiotics (of different types and/orconcentrations). After incubation, zones of microbial growth inhibition114 are formed. These zones 114 are interpreted to indicate resistant,intermediate or susceptible microorganisms based on NCCLS criteria.

Another method of antimicrobial susceptibility testing is the antibioticgradient method. This test utilizes an antibiotic gradient in a gelmedium. Paper or plastic strips are impregnated with an antibioticconcentration gradient. A plurality of strips is placed on aMueller-Hinton agar plate like spokes on a wheel, with the plate havingbeen inoculated with the microorganism to be tested. After incubation,an antibiotic gradient is formed in the gel in an elliptical shapearound each test strip (if the microorganism is susceptible to theantibiotic on the particular strip). The minimum concentration of theantimicrobial agent that prevents visible microorganism growth is theendpoint of the test (the minimum inhibitory concentration, or MIC). Putin other words, in disk diffusion testing, the MIC is the concentrationat the edge of the inhibition zone (the growth/no growth boundary). Inthis case, the MIC is the point at which the elliptical growthinhibition area intersects the test strip. As can be seen in FIG. 15,agar plate 101 has a plurality of test strips 103 that are impregnatedwith an antibiotic gradient. Elliptical zones 105 are formed wheremicroorganism growth is inhibited by the antibiotic agent in/on the teststrip. Point 107 where the elliptical zone intersects the test strip isthe MIC point.

A third type of susceptibility test is the broth dilution test. In thistype of test, dilutions of antibiotics (e.g. consecutive two-folddilutions) are prepared. Often, at least ten concentrations of a drugare prepared in tubes or microwells. Each tube or well having thevarious concentrations of antibiotics is inoculated with a particularmicroorganism (a standardized suspension of test bacteria is added toeach dilution to obtain a final concentration of 5×10⁵ CFU/ml). A growthcontrol well and an uninoculated control well are included on eachplate. After incubation (e.g. for 16-24 hours at 35 degrees C.), thewells or tubes are examined manually or by machine for turbidity, hazeand/or pellet. Indicators can be placed in the wells to facilitate thevisualization of microbial growth. As with other tests, the minimumconcentration of antimicrobial agent that prevents visible microbialgrowth is the MIC.

Commercial microdilution tests are typically performed on standard 96well plates, each well holding approximately 100 to 200 microliters withcommercially prepared antibiotic test panels. With 96 wells and 2 to 10different dilutions for each antibiotic, numerous antibiotics can betested on a single plate. A significant problem with such commercialmicrodilution systems is the inflexibility of the standard antibiotictest panels. The commercial plates are manufactured with various amountsof frozen, dried or lyophilized antimicrobial agents in the wells. Thisavoids the time consuming task of preparing the plates. However, due tothe availability of many antibiotics (more than fifty in the UnitedStates), it is often problematic for a laboratory to find a standardcommercial test panel which is ideal for that laboratory's needs. FIG.16 is an illustration of a 96-well plate used in such a microdilutionsystem.

A variation of the broth microdilution method is set forth in U.S. Pat.No. 5,501,959. This system uses microtiter plates with 168 wells, eachcontaining a paper disk attached to the bottom of the well. The diskscontain serial two-fold dilution concentrations of various antimicrobialagents, as well as a redox indicator. Up to 20 different antimicrobialagents can be tested on a plate. This use of paper disks simplifies themanufacture of the custom panels. However, higher costs are involvedwhen a susceptibility test is custom made for a customer.

Current instruments that offer the highest degree of automation insusceptibility testing are typically based on automating the tasksperformed in the manual broth microdilution method mentioned above. Onesuch example is the instrument described in U.S. Pat. No. 4,448,534.This instrument uses multi-well plates that are pre-loaded with serialtwo-fold dilution concentrations of antimicrobial agents. Plates areinoculated manually and placed in the instrument, where they areincubated. At the appropriate times, the wells on the plate are read bya photometer/fluorometer to determine the results of the test. Anotherautomated system is described in U.S. Pat. No. 3,957,583. Thisinstrument uses small multi-chamber cards that are pre-loaded withserial two-fold dilution concentrations of antimicrobial agents. Cardsare inoculated automatically, incubated, and monitored within theinstrument. This instrument reads the chambers in the card periodicallyusing a photometer. These kinetic measurements yield growth curves thatallow the instrument to determine the results of the test. Though theaforementioned instruments perform testing in 4 to 8 hours, they mayfail to detect induced resistance of the microorganism, which couldresult in an incorrect susceptibility report Unfortunately, the degreeof automation that is provided by instruments based on brothmicrodilution is not available for methods such as disk diffusion.

Regarding microbial identification, various selective and differentialmedia have been relied upon for determining which type of microorganismhas been detected. Selective media are appropriate when testing forspecific genera or species of microorganisms and act by inhibiting all(or nearly all) microorganisms except the target microorganism.Differential media are used to distinguish between certain species ofbacteria based on a particular trait (e.g. the ability to metabolizecitrate as a sole carbon/energy source). In a hospital or environmentalsetting, the different species one might encounter are of such anoverwhelming number, that the use of selective media is prohibitive.Microbial identification in these settings is then based on metaboliccharacteristics. Conventional techniques require a large time commitmentfor the preparation of the appropriate media and much laboratory spaceusing traditional equipment.

There are also a variety of automated instruments that exist foridentification of microorganisms. In one such instrument, smallmulti-chambered cards contain a variety of substrates. The chambers aremeasured photometrically. Based on the pattern of substratesmetabolized, identification is determined. Another system uses a 96 wellplate. Each well contains an individual carbon source and red-oxindicator. Identification is determined based on the pattern ofsubstrate utilization. Still another system uses fluorescent dyes andsubstrates labeled with fluorescent compounds. Enzymatic activityreleases the fluorescent compound from these substrates or changes inthe pH result in a change in the intensity of the fluorescence.

SUMMARY OF THE INVENTION

The present invention relates to detection and/or screening ofmicroorganisms on an instrument, and concurrently or consecutively onthe same instrument, determining the susceptibility of microorganisms tovarious antibiotics and/or identifying microorganisms. The inventionalso relates to optionally performing urine screening within the sameinstrument. The instrument employs image acquisition technology, severalimage processing algorithms, and a variety of specialized disposableplates to perform these functions.

More particularly, the instrument employs disposable plates forculturing microbial organisms, and computer hardware and software tofacilitate detection and/or measurement by the instrument. Because thesystem performs a variety of functions, there are several types ofspecialized plates that are used within the instrument. Each type ofplate is optimally configured for its particular function; hence, theplates may differ significantly in terms of culturing and detectionproperties, and physical dimensions. However, all plates, regardless oftype, can be processed by the instrument The instrument is capable ofperforming multiple measurements on each plate over time and utilizingthis kinetic rate information to more accurately perform each function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of the main components of the multi-functionalinstrument;

FIG. 2 is an illustration of one embodiment of the invention showingdifferent types of plates, imaging devices, and transport systems;

FIG. 3 is an illustration of another embodiment of the invention showingplates, plate shelves, imaging/lighting devices and transportmechanisms;

FIG. 4 is an illustration of a cross section of the embodiment shown inFIG. 3;

FIG. 5 is an illustration of an additional embodiment of the instrumentof the invention;

FIG. 6 is an illustration of a cross section of the embodimentillustrated in FIG. 5;

FIG. 7 is an illustration of yet another embodiment of the instrument ofthe invention;

FIG. 8 is an illustration of a cross section of the embodimentillustrated in FIG. 7;

FIG. 9 is an illustration of a microbial detection (sensor) plate;

FIG. 10 is a cross section of the sensor plate;

FIG. 11 is a cross section of an alternate embodiment of the sensorplate;

FIG. 12 is a cross section of a further embodiment of the sensor plate;

FIG. 13 shows the bottom of three sensor plates positive for E. coli;

FIG. 14 is an illustration of an agar plate for performing a diskdiffusion antibiotic susceptibility test;

FIG. 15 is an illustration of an antibiotic gradient method fordetermining susceptibility of a microorganism to particularantimicrobial agents;

FIG. 16 is an illustration of a device for performing a brothmicrodilution antibiotic susceptibility test;

FIG. 17 is an illustration of one embodiment of the present inventionhaving a bottom portion with a bottom gel plate having internalpartitions, and a top cover;

FIGS. 18a and 18 b are top views of two embodiments of the inventionwhere FIG. 18a illustrates elongated channels with antibiotic disks, andwhere FIG. 18b illustrates shorter channels, some with antibiotic diskstherein;

FIG. 19a illustrates an embodiment with elongated channels each havingan antibiotic strip therein, whereas FIG. 19b illustrates an embodimenthaving thin shorter channels with antibiotic disks at one end thereof;

FIGS. 20a and 20 b are views of a susceptibility plate with E. coliwhere FIG. 20a shows a raw image and FIG. 20b shows a processed image;

FIGS. 21a and 21 b are views of a susceptibility plate with S. aureus,where FIG. 21a shows a raw image and FIG. 21b shows a processed image;

FIGS. 22a to 22 d are additional views of a susceptibility plate forKleb. pneumoniae, where FIG. 22a is an unprocessed image after 4 hours,FIG. 22b is a processed image after 4 hours, FIG. 22c is an unprocessedimage after 18 hours, and FIG. 22d is a processed image after 18 hours;

FIGS. 23a and 23 b are views of an identification plate, where FIG. 23ais a plate viewed at a wavelength of visible light after 24 hours, andFIG. 23b is a plate viewed in the ultraviolet range;

FIG. 24 is another view of the identification plate of the invention;and

FIGS. 25a and 25 b are views of urine screening plates (unprocessedimages) taken at 18 hours.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As can be seen in FIG. 1, a processing subsystem 200 is provided incommunication with a user interface subsystem 202, a plate transportsubsystem 204, incubation subsystem 206 and imaging subsystem 208. Theprocessing subsystem is responsible for controlling the operation of theinstrument, and is based on an embedded microprocessor (e.g., Pentium™)or microcontroller board or system, such as a PC. Using various parallelor serial interfaces, the processing subsystem communicates with, aswell as controls, the other subsystems. The processing subsystem alsoprovides the capability to perform analysis of acquired data. The userinterface subsystem provides the capability for users to control theinstrument, as well as view control parameters and/or data from theinstrument. The user interface subsystem is comprised of a CRT, LCD,CCD, and/or CMOS display, coupled with discrete buttons, a barcodereader, a keypad, a keyboard, and/or a touchscreen. Audio input (withspeech recognition) and output capabilities may also be provided. Theincubation subsystem provides the capability for regulating temperatureand/or humidity within the incubation chamber of the instrument, whilethe plate transport subsystem provides the capability to control themovement of devices within the instrument. The imaging subsystemprovides the capability to acquire images of the plates, and comprisesvarious image capture and illumination devices.

Generally, the instrument is responsible for performing three mainfunctions (though additional functions are envisioned): plateincubation, image acquisition, and image processing. The instrumentprovides a controlled environment to incubate the various plates foroptimal growth conditions. The plates (detection/enumeration sensorplates, antibiotic susceptibility plates, identification plates, urinescreening plates, etc.) are inoculated and placed in the incubator wherethey are subsequently scanned at regular intervals by an imageacquisition device during the incubation period. The instrument providesfor image acquisition using one or more color and/or gray scale imagingand illumination devices. The time-lapsed images are obtained from oneor more sides (views and angles) of the plates. Images acquired duringthe incubation period are analyzed using several image processingtechniques and algorithms to determine results. The instrument designcan be modular such that it can be configured to provide a wide range ofplate incubation capacity.

One example of how the variety of plates could be moved relative to oneor more imaging devices is illustrated in FIG. 2. As can be seen in thisfigure, imaging devices 211 and 212 are provided for capturing the imageof the top and bottom, respectively, of each plate. Top and bottomimaging transport systems 215 and 216 are provided for moving therespective imaging devices past a plurality of plates when the platesare disposed at the top or bottom imaging station. Each plate 217 ateach imaging station has its image captured by an imaging device. Platesare moved to the imaging stations by a plate transport system 218 that,as in FIG. 2, moves plates both upwardly and downwardly from the imagingstations. In one possible configuration, plate shelves holding theplates are transported down the front side of the instrument and up therear side. The imaging devices are mounted on linear tracks at the topand bottom positions of the rotating track. The plate shelves arerotated “stepwise” within the instrument. The plate shelves at the topand bottom positions of the rotating track enter “imaging stations”. Theimaging devices are tracked along the length of the shelves, capturingimage data. Plate shelves are then rotated another “step”, and othershelves are moved into the imaging locations for image capture. Thoughthe different types of plates are illustrated as being the same size,the plate holders can also be constructed to hold plates of varyingsizes, or different plate holders could each hold plates of a particularsize, with plate sizes varying between plate holders.

In another embodiment of the invention, as can be seen FIGS. 3 and 4, aplurality of plates 220 are held on plate shelves 221. The plate shelvesare “stacked” only on the front side of the instrument. Shelves aremounted on slide bearings that allow them to slide back into theinstrument for imaging. The imaging/lighting devices 223 are mounted onan imaging/lighting transport mechanism 225, such as an X-Y trackingmechanism behind the shelves. Plates are imaged in the following manner:

a) The imaging devices are moved into position (aligned with the levelof the shelf) behind the shelf;

b) The shelf is pulled back into the instrument along linear bearings,between imaging devices;

c) The imaging devices are tracked along the length of the shelf,capturing image data;

d) The shelf is pushed forward, and the imaging devices are moved toimage the next shelf.

In a further embodiment of the invention as can be seen in FIGS. 5 and6, plates 230 are held on plate shelves 232 (the plate shelves can beconfigured in the same manner as in FIGS. 3 and 4). The imaging devices234, are provided as long scanner arrays arranged along the length ofthe shelves. Plates are imaged in the following manner:

a) The scanner arrays are moved into position by a scanner transportmechanism 236 (aligned with the level of the shelf) behind the shelf;

b) The shelf is pulled back into the instrument along linear bearings,between the scanner arrays;

c) As the shelf is pulled back, all of the plates on this shelf areimaged simultaneously by the array of scanners; and

d) The shelf is pushed forward, and the scanner arrays are moved toimage the next shelf.

In yet another embodiment of the invention, as illustrated in FIGS. 7and 8, plate shelves 243 with plates 240 thereon are moved within theinstrument on a rotating track (as in FIG. 2). As in FIG. 5, the imagingdevices 245 are scanner arrays arranged along the length of theshelves—however these arrays are fixed at the top of the instrument.Plates 240 are imaged in the following manner:

a) Plate shelves are rotated “stepwise” within the instrument;

b) As the shelves are moved from the back of the instrument towards thefront (at the top of the rotating rack), they pass between the scannerarrays (mounted at the top of the instrument) where all plates areimaged simultaneously; and

c) Plate shelves are rotated another “step”, and another shelf is movedthrough the scanner arrays for image capture.

Of course other plate and/or imaging device transport systems could beutilized. A cartridge system could be used, where one plate at a time isremoved from a stack for imaging (and either the stack or imaging deviceis moved to position the imaging device and selected plate in proximityto each other). A turntable or other rotary system could be used toposition each plate proximate to an imaging device, or a conveyor beltor wheel system could be used, where plates (with solid media) areaffixed to a belt or wheel in rows and are turned upside down dependingupon which side of the belt or wheel the plate is on.

The instrument provides for image acquisition using one or more colorand/or gray scale imaging devices: CCD linear array scanner, a CCDline-scan camera, a CCD 2D array camera (still or motion video), a laserscanning camera, or other device that would provide a sufficiently clearimage of the plate that can be used alone or after further processing.By “image” it is meant any information, such as optical information,from the plate that is ≧a 1×1 pixel. The image acquisition is performedat regular pre-programmed intervals, with the captured image obtainedfrom one or more views and angles of the plate.

Images acquired during the incubation period are analyzed using one ormore image processing techniques. In the example of FIG. 2, images areacquired from both the top and bottom of the plate at regular intervals(though imaging only one side of the plate is also envisioned).Typically the interval is from 5 minutes to 4 hours, though preferablythe interval is between one half-hour and 2 hours, and most preferablyevery hour. Scanning at regular intervals provides kinetic growth data,which may be used to help characterize the microorganisms. Theimage-processing algorithm implemented to determine results on eachplate is comprised of one or more of the following steps:

a) Color Separation and Selection (if required)—to select the color bandof interest (sensor dependent);

b) Image Masking—to isolate the area of interest from extraneous imagedata;

c) Image Subtraction—to isolate the areas of change between two imagestaken at subsequent time intervals;

d) Image Equalization—to amplify the magnitude of the changes appearingin the subtracted image;

e) Image Blurring—to reduce the effects of single pixel noise in theequalized image (e.g. low pass filter);

f) Image Contrast and Brightness Enhancement—to further amplifylocalized differences in the filtered image;

g) Image Thresholding (with several thresholds, if required)—to preparethe image for the colony detection/enumeration algorithm;

h) Processed Image Analysis using one of the following (or other)functions depending on the plate being analyzed—

1) Colony Detection, Enumeration and/or Classification—to determine thepresence of microbial organisms on the plate, to enumerate the number ofcolonies on the plate, and/or to perform color analysis to classifycolonies on the plate; or

2) Inhibition Zone Detection and Measurement—to determine thesusceptibility of microbial organisms to a particular antibiotic; or

3) Substrate Reaction Zone Inspection—to determine the extent ofsubstrate reaction by color analysis.

Plates can be illuminated by a separate lighting mechanism, or the lightsource can be provided as an integral part of the imaging device. Thelight source can be broadband and/or narrowband visible, ultravioletand/or infrared illumination.

As will be explained below, a variety of plates can be handled andanalyzed, all within the same instrument:

Detection/Enumeration Plate:

FIG. 9 is an illustration of sensor plate 1 that can be in the form of aflat, shallow container with at least one side (e.g. the bottom side)being transparent or translucent Though the container can be open (oreven simply a substrate), it is preferably a sealed or sealablecontainer, and preferably with an amount of headspace above the sensorplate layers. The container can be provided with a port 2, which may besealed with a stopper 3, screw-cap, septum, or any combination thereof(or any other sealing device). Once a sample is collected into thecontainer, the sensor plate can be configured as either a gas-permeableor a gas-impermeable container, depending on the growth requirements ofthe microorganism. This configuration is accomplished by using differentplate composition materials, laminates (gas impermeable and/orhydrophobic gas-permeable membranes), and/or configurable vents (eg. agas permeable membrane in an opening of the container wall).

Within the container of the sensor plate device, are one or more layerswhich help to immobilize/absorb the sample so that colonies ofmicroorganisms can grow localized which increases the ability to detectthe colonies of microorganisms. In one embodiment, at least one layer inthe device has matrixes that adversely affect visualization ofmicroorganisms. As can be seen in FIG. 10, provided are an immobilizinglayer (matrix layer) 10 and a sensor layer 12. These two layers, whichwill be described more fully hereinafter, can also be combined togetherinto a single layer, though it is preferred that the two layers beprovided separately. As also shown in FIG. 10, is the plate bottom 14,which is preferably transparent for viewing/imaging changes in thesensor layer due to microorganism growth.

The sensor layer 12 is provided for the purpose of indicating thelocation of microbial growth by providing a tightly localized dramaticchange in the ultraviolet, visible, and/or infrared spectrum. Thislocalized change is detectable on the bottom surface of the plate,opposite the sensor surface near the microbial growth. The sensor layercomprises a material that undergoes a change in a detectable property(e.g. an indicator) which is embedded on and/or in a matrix (supportmaterial) which is preferably opaque. By “opaque”, it is meant that thesensor layer sufficiently blocks the viewing or detecting (in anyrelevant electromagnetic region) of the test sample and/or actualmicroorganism colonies immobilized in the immobilization layer from theopposite side of the sensor layer (e.g. semi-opaque, substantiallyopaque, or fully opaque). Though it is possible to have a transparent orrelatively transparent sensor layer if the test sample is alsosubstantially transparent (in which case the sensor layer undergoeslocalized changes from transparent to opaque in the presence ofmicroorganism colonies), it is preferred that the sensor layer not betransparent. Improved results are obtained in detecting microorganismsin test samples that could interfere with detection and enumeration ifthe sensor layer is opaque. If the test sample itself interferes withvisualizing/detecting (e.g. with the eye or with an instrument) thepresence or growth of microorganisms directly in the immobilizationlayer, then it is preferable that at least one of the immobilizationlayer or the sensor layer (preferably the sensor layer) is capable ofblocking detection/visualization of the actual test sample and/or actualmicroorganisms, and instead detect changes in the sensor layer whichcorrespond to presence/growth of microorganisms in the immobilizationlayer. The immobilization layer can also be opaque, and in oneembodiment of carrying out the invention, the sensor layer, theimmobilization layer, and the sample are all opaque.

The sensor comprises a solid composition or membrane, with an indicatormedium immobilized on or within it The sensor layer is preferablylocated flush against the inside surface of the container, or in thesealing means used to seal the container or attached to the sealingmeans, such that the sensor layer is visible from outside. It ispreferably affixed to the container to prevent cells, proteins, othersolids or other opaque or colored components from getting between it andthe container surface. In certain embodiments the sensor layer isseparated from the specimen and its growth medium by a membrane or othersolid layer.

One embodiment of this invention comprises a sealing means, such as alid or cap, which may be transparent or which may have a transparentsection. The sensor can be placed in proximity to the transparent lid orsection of lid or is made part of the lid. When the lid is used to sealthe container, the changes in indicator are read through the transparentsealing means. The sealing means may also be made of a material, such asa polymer, which contains encapsulated indicator micelles. A transparentsection in either the container or the sealing means is not needed, aslong as the material is permeable to the changes caused by metabolism ofthe microorganisms, and the changes in the indicator are visible on thesurface of the sealing means.

Microorganisms in specimens of body fluids, such as blood, containing asfew as 1 organism per total sample volume, can be detected using thisinvention. Such specimens may require a number of days of incubationbefore the population of organisms reaches a critical level and where achange in a parameter involved in microorganism metabolism can bemeasured.

The sensor is useful in that: 1) changes in the sensor layer due tomicrobial metabolism (e.g., increases or decreases in a gas componentdue to metabolism) are detected from the solid or semi-solidimmobilizing layer rather than in the atmosphere over the specimen, 2)because the sensor is affixed to the interior surface of the plate orthe closure or sealing means or attached through the outside of theclosure or sealing means, measurements can be made from outside thetransparent wall of the plate or the sealing means without having toviolate the integrity of the plate, 3) the external measurements can bemade by visual inspection or with an instrument that measures byreflectance, fluorescence, etc., or by image capture, 4) opaque/coloredor fluorescent components in the specimen do not interfere with theability to detect changes or the measurement of those changes, and 5) ahigh concentration of indicator molecules can be maintained within asmall volume in the sensor (e.g., within the polymer emulsion or on themembrane), such that a change can be easily observed or detected.

The nutritional components that make up a complex microbial mediuminfluence the metabolic pathways used by microorganisms. Organic adds,bases and various gases are produced in proportions dependent on thenutrients available. These products also vary from species to species ofmicroorganism. The presence of these products in the immobilizing layercan change its pH. The sensor layer used in the invention could containpH sensitive indicators that give a measurable change in response to apH change. Or, the presence of gases that affect the pH of theindicator, such as CO₂, could be measured. Microbial growth can also bedetected by measurement of changes in O₂ and/or fluorescence. The sensorlayer can be designed to respond to decreases in O₂ concentration due tometabolism of microorganisms. And an indicator could be selected thatundergoes a change in fluorescence rather than a change in color orother parameter. Carbon dioxide is a common metabolite produced by mostorganisms and, therefore, is the preferred metabolite for detection ofmicrobial growth. Whatever mechanism is utilized, in a preferredembodiment, the sensor layer will undergo a detectable change inresponse to the presence growth of most microorganisms.

The indicator can be attached either covalently or non-covalently to asupport medium. Alternately, the indicator can be encapsulated within apolymer matrix such as being emulsified within a polymer matrix prior tocuring.

The sensor layer is preferably affixed inside a suitable transparentvessel or a transparent sealing means, with an appropriate adhesive, ifnecessary. They may also comprise an integral part of the sealing meansor be affixed to the sealing means or within the vessel as an indicatoremulsified within a polymer matrix cured in situ. They can also beplaced outside the container, as long as a method is provided thatallows the metabolic changes due to the microorganisms, to affect thesensor.

A variety of different fluorescent and visible pH indicators can be usedas the active molecular species in pH, H₂, H₂S, NH₃, O₂ or CO₂ sensors.Generally, the only limitations on the selection of indicators are therequirements that they have acceptable dynamic ranges and wavelengthchanges that are detectable by infrared, fluorescence, reflectanceand/or imaging technologies.

Sensors for detecting pH changes in the culture medium according to theinvention preferably exhibit a change in fluorescence intensity orvisible color over a pH range of about 5.0 to about 8.0.

Indicators for a CO₂ sensor should exhibit a change in infraredintensity, fluorescence intensity or visible color preferably betweenabout pH 13 and about 5, and most preferably between about pH 13 toabout 9, in order to detect changes in CO₂ concentration.

Only certain pH indicator molecules can be bound covalently ornon-covalently to a support medium and retain their pH indicatingproperties. Indicators belonging to the xanthene, phenolphthalein andphenolsulfonphthalein groups are useful. Examples of these includefluorescein, coumarin, phenolphthalein, thymolphthalein, bromothymolblue, thymol blue, xylenol blue, ortho cresolphthalein and α-naphtholbenzein.

The support medium can be a substance such as cellulose or certainsilicones, to which a pH indicator can be covalently attached usingorganic reactions. Non-covalent attachment of pH indicators can beachieved using ionic support materials, such as nylon membranes thathave a positive or negative zeta potential. Other ionic supportmaterials that can be used are positive or negatively charged ionicresins, such as diethylamino ethyl (DEAE) resin or DEAE cellulose.Pretreatment of the support material with a protein may be required ifthe indicator membrane is to be in direct contact with microbial growthmedium.

The pH indicator sensors directly detect pH changes due to the pHenvironment of the microbial growth medium. However, these sensors canbe made to selectively react to gases (e.g., carbon dioxide, ammonia,hydrogen, hydrogen sulfide, or oxygen) due to microorganism metabolism.A selectively semi-permeable composition or membrane could be providedon the sensor layer, such as silicone, latex, teflon, or variousplastics characterized by the capacity to selectively permit thediffusion of a gas while preventing the passage of ions. For sensorscomprising indicator encapsulated within a polymer matrix, the polymerforming the matrix can act as the semi-permeable barrier that permitsthe passage of gases but not ions.

In one embodiment, the CO₂ sensor is comprised of a plurality ofcomponents. The first component is a visual or fluorescent pH indicator,which is reactive at the pH range between 6 and 10. Examples ofindicators meeting these criteria are bromothymol blue, thymol blue,xylenol blue, phenolphthalein, ortho cresolphthalein, coumarin, andfluorescein. A second component, if necessary, is an acid, base orbuffer, which maintains an optimal pH environment for detection of CO₂by the selected pH indicator. A third component can be glycerol or anequivalent emulsifier, which can produce droplets of indicator solutionemulsified within the uncured polymer. A fourth component can be apigment, such as titanium oxide, zinc oxide, magnesium oxide, ferrousoxide, etc. A fifth component can be an uncured polymer such assilicone, which maintains a proper environment for the indicator. Anypolymer can be used that does not affect too greatly the chemicalactivity of the indicator, either from its own chemical or physicalproperties or its requirements for curing, as long as it is permeable togases but not ions, and does not have these properties altered whensubjected to sterilization. Other silicone polymers that are alsosatisfactory are those that are cured by high temperature, by catalyticactivity, or by ultraviolet vulcanization. An emulsion is prepared fromthe various components and the polymer is cured to form a semipermeablematrix around the droplets of pH indicator, which permits selectivediffusion of CO₂ and other gases from the immobilization layer,resulting in localized measurable changes in the sensor layer. Thesensor layer can be prepared separately, such as in a mold, cured, andthen attached to the plate with an appropriate adhesive, such as asilicone adhesive. Alternatively, and preferably, the sensor is formedon the bottom of the container and cured in situ. After curing, thecontainer with the sensor can be sterilized, such as by autoclaving orgamma radiation. Conveniently, the immobilizing and additional optionallayers can be introduced into the sensor plate device beforesterilization and thus also sterilized by that process.

In a further example, the sensor layer comprises an indicator solutionemulsified in a pigmented silicone matrix. The indicator solution iscomprised of thymol blue indicator (0.65 g) dissolved into a solution of0.8 M potassium hydroxide (10.0 ml) and isopropyl alcohol (10.0 ml). Theindicator solution (5.0 g) is then mixed with the pigmented siliconecomponents. The pigmented silicone matrix is comprised of Sylgard 184silicone (components A (50.0 g) and B (5.0 g)) and white pigment (part#61-18000, Ferro Corp., New Jersey) (1.0 g). The sensor material is thenpoured and spread onto a plate in a thin layer (approximately 0.2 to 0.5mm).

In another example, the sensor layer comprises an indicator solutionmixed with a pigmented silicone matrix. The indicator solution iscomprised of ortho-cresolphthalein indicator (2.0 g) dissolved into asolution of isopropyl alcohol (5.0 ml) and 0.9 M potassium hydroxide(5.0 ml). The indicator solution (2.5 g) is then mixed with thepigmented silicone components. The pigmented silicone matrix iscomprised of Sylgard 184 silicone (components A (25.0 g) and B (2.5 g))and white pigment (part #61-18000, Ferro Corp., New Jersey) (0.5 g). Thesensor material is then poured and spread onto a plate in a thin layer(approximately 0.2 to 0.5 mm). In a variation of this example, the aboveortho-cresolphthalein sensor layer is covered with an overcoat layercomprising the pigmented silicone matrix.

In still another example, the sensor layer is composed of an indicatorsolution mixed with a pigment solution and a silicone matrix. Theindicator solution is comprised of ortho-cresolphthalein indicator (2.0g) dissolved into a solution of isopropyl alcohol (10.0 ml), and 0.8 Mpotassium hydroxide (10.0 ml). The pigment solution is comprised ofsilicone oil (40.0 g), white pigment (part #61-18000, Ferro Corp., NewJersey) (4.0 g). The silicone matrix is comprised of Wacker Elasbosil RT601 silicone (components A (200.0 g) and B (20.0 g)) and toluene (40.0g). The indicator solution (20.0 g) is then mixed with the pigmentsolution (40.0 g) and silicone components. The sensor material is thensprayed onto a plate in a thin layer (approximately 0.1 to 0.3 mmthick).

In addition to indicators responsive to changes in oxygen, carbondioxide and pH, as mentioned above, indicators could also be utilizedthat detect changes in ammonia, oxidation-reduction potential, hydrogen,hydrogen-sulfide, or any other substance that undergoes a change due tothe presence or growth of microorganisms. Also, a plurality of differentindicators could be used in the sensor layer (or in a plurality ofsensor layers).

The sensor layer is preferably opaque so as to prevent properties of thesample (e.g. natural fluorescence, opacity, etc.) from affecting ormasking the response of the sensor. The sensor layer preferably changesfrom one opaque state to another opaque state in the presence ofmicroorganisms, with the change being a detectable change by imagecapture and processing. As one example, the sensor layer could be anemulsified mixture of ortho cresolphthalein indicator in a whitepigmented silicone matrix, with an overlay of white pigmented silicone.Or, the sensor layer could be a pigmented silicone matrix emulsifiedwith one or more indicators such as thymol blue indicator, a xylenolblue indicator, or a “universal” indicator. The matrix in the sensorlayer could be a suitable latex, polyurethane, nylon membrane (e.g.charged nylon membrane) or cellulose powder. The sensor layer matrixcould also be a silicone matrix, such as Sylgard 184, Wacker 601, orWacker 934. Or, the sensor layer could be made up of two layers, such asan indicator layer and an opaque layer.

The other main layer in the sensor plate device is the immobilizinglayer 10. The purpose of the immobilizing layer is to immobilizeorganisms in the sample either within a matrix or on the surface of amatrix. The sample itself can be a liquid, semi-solid or semi-liquid(e.g. paste or gel) sample. A liquid sample can be mixed with a drypowdered gelling agent to form an organism-immobilizing gel matrix whenmixed. The liquid sample is preferably added to a dry powdered gellingagent already provided as a layer in the sensor plate device. However, aliquid sample could also be mixed with a dry powdered gelling agent, andthen both immediately added to the sensor plate device before gellinghas occurred. Also, a liquid sample could be applied onto an alreadygelled matrix, or onto a dehydrated or partially dehydrated gel matrixso as to immobilize the microorganisms on the surface of the gel. Agelling agent could also be imbedded in a support matrix to add physicalsupport. Examples include glass or cellulose synthetic polymer fiberseither mixed throughout or in the form of woven or non-woven fabrics.Also, in order to immobilize a sample, the immobilization layer need notbe a gelling agent, but rather could comprise a non-gel absorbentmaterial, such as sponge materials, celluloses, glass fibers, filterpaper, etc.

More than one gelling agent could be utilized in the sensor platedevice, either mixed together or as separate layers. For example, amixture of guar gum and xanthan gum, combined by weight at anapproximate ratio of 2:1, could be used. Other gelling agents could beused singly or in combination, including natural and synthetic hydrogelpowders. One or more gelling agents could be combined together selectedfrom gums, agars, agaroses, carageenans, bentonite, alginates,collagens, gelatins, fused silicates, water soluble starches,polyacrylates, celluloses, cellulose derivatives, polyethylene glycols,polyethylene oxides, polyvinyl alcohols, dextrans, polyacrylamides,polysaccharides or any other gelling or viscosity enhancing agents.

Dehydrated and/or partially dehydrated gel matrices for surface colonyisolation/immobilization could be used, including one or more syntheticor natural hydrophilic polymers. If more than one gelling agent is used,such could be mixed together or provided in a plurality of layers. Inone example, an upper layer could be provided to trap microorganisms onthe surface, and a lower layer could be provided as a wicking agent todraw the liquid sample through the upper layer (e.g. a thin agar layerover a modified cellulose absorbent, or a porous hydrophilic membraneover an absorbent pad, polymer or hydrogel).

The immobilization layer must not adversely affect the sensor layer. Ifthe sensor layer undergoes a detectable change due to a pH change, thena very acidic gel layer could adversely affect the sensor layer (alsosome manufacturing processes are acidic and could leave an add residuethat could adversely affect the sensor layer). Furthermore, if theimmobilization layer is a powdered gel layer, it should be certain thatthis layer does not turn acidic when mixed with a sample, as this couldalso cause the sensor layer to change even in the absence ofmicroorganisms.

As can further be seen in FIG. 10, an optional conditioning layer 16 canbe provided on (or within or below) the immobilizing layer. Thoughillustrated separate from the immobilization layer in FIG. 10, theconditioning materials from the conditioning layer are preferablyincorporated into the immobilization layer itself. Conditioningcomponents, whether provided within the immobilization layer or in aseparate layer, can include one or more of media for microorganismgrowth, lytic agents, lytic enzymes, antibiotic neutralizers,surfactants or other materials helpful for improving microorganismdetection and/or enumeration capabilities. Conditioning components canalso be provided both within the immobilization layer and in a separatelayer in the same sensor plate device.

Lytic agents for conditioning can be added for lysing blood cells in thetest sample, for allowing for a smoother gel, and/or for betterrehydration of the gel. Examples of possible lytic agents includesaponin, digitonin, Tweens™, polysorbitan monolaurate, and othersurfactants. Lytic enzymes, typically tough not necessarily proteolyticenzymes, may be added for digesting cellular material in a blood sample,for making a smoother gel, and/or for better rehydration of the gel. Thelytic enzymes for conditioning can include one or more proteases, forexample an enzyme mixture derived from Aspergillus oryzae, or the like.

Antibiotic neutralizers may be added for conditioning, in particular forfaster and/or better recovery of microorganisms in the test sample. Oneor more of such neutralizers could be selected from resins, gums, andcarbon-based materials (e.g. activated charcoal or Ecosorb™), or one ofa variety of enzymes to specifically degrade various antibiotics (e.g.beta lactamase).

Media can also be added for conditioning (whether directly to theimmobilization layer or separately). Media is added to provide nutrientsfor the growth of microorganisms. Though many types of media fordifferent types of microorganisms could be used, if the microorganism isan aerobic organism, the media could include, as one example (anexemplary amount of each being listed in parentheses in g/l): tryptone(17), soytone (3), protease peptone (5), malt extract (2.5), dextrose(2.5) and MOPS (23). If the microorganism is an anaerobic organism, themedia could further include the media listed above for aerobicorganisms, as well as Hemin (0.005), L-cystine (0.2), Na-m-bisulfide(0.2) and Menadione (0.0005).

For Coliforms, the media could include, as an example, Lactose (5), bilesalts #3 (0.8), K₂HPO₄ (7), KH₂PO₄ (3), (NH₄)₂SO₄ (0.5), MgSO₄ (0.1),Na-m-bisulfide (0.4) and SDS (0.1). For yeast, mold and other addtolerant microorganisms, the media could include, as one example,dextrose (10), yeast extract (10), (NH₄) citrate (2) and tartaric acidto a pH of 5.5.

As can be further seen in FIG. 10, a wall of the container can beprovided with apertures 20, below which is a hydrophobic gas-permeablefilm 22, and above which is a gas-impermeable (removable) film 24. Or,the container could be provided with an opening in a wall thereof withthe gas-impermeable film and the hydrophobic gas-permeable film adheredtogether covering the opening. If the organism is anaerobic, thegas-impermeable film would be left in place. However, if the organism isaerobic, the gas-impermeable film would be removed at the time of theaddition of a test sample to the sensor plate device. Of course, thehydrophobic gas-permeable film need not be provided at all, though it isbeneficial for preventing contaminants from entering the container, andfor preventing potentially infectious test material from leaking out ofthe device.

Area A in FIG. 10 is illustrated in further detail in FIGS. 11 and 12.As can be seen in FIG. 11, in a further embodiment of the sensor platedevice, in place of a single immobilization matrix layer, there can beprovided one or more of: an isolation gel layer 30 for a semi-rigidsurface to allow surface capture and recovery after growth, an adhesivelayer 31, an absorptive gel layer 32 and an additional adhesive layer33. The absorptive gel layer 32 can include one or more of conditioningcomponents (in gels), media for microorganism growth, lytic enzymes, andantibiotic neutralizers. As can be further seen in FIG. 11, in place ofa single sensor layer, there can be provided one or more of: an overcoatlayer 34, an adhesive layer 35, an indicator layer 36, and an additionaladhesive layer 37 in contact with plate bottom 38.

In an additional embodiment of the invention as illustrated in FIG. 12,provided is a matrix layer 40 which comprises: a gelling powder, and dryconditioning components such as media, lytic enzymes and antibioticneutralizers. As in FIG. 11, in place of a single sensor layer, therecan be provided one or more of: an adhesive layer 41, an overcoat layer42, an adhesive layer 43, an indicator layer 44, and an adhesive layer45 in contact with plate bottom 46.

The size of the sensor plate device can be varied depending upon thedesired sample size. In one example, a sensor plate device has animmobilization layer of the dimensions of 74 mm×117 mm. If theimmobilization layer comprises a wet-type gel, then the sample sizecould be made very small (e.g. 1 ml or less), or, such as with a bloodsample, the sample size could be up to 15 ml. On the other hand, if theimmobilization layer comprises a dry powdered gel, then the sample sizecould be even greater, depending upon the amount of the powdered gel(e.g. the sample could be 30 ml or more).

In use, a fluid sample is introduced into the sensor plate device. Thesample is “conditioned” (if desired) as it spreads across the bottomsurface of the sensor plate. The sample is absorbed into, or forms a gelwith, an immobilization matrix layer. The sensor plate is thenincubated, promoting the growth of microorganism colonies. A sensorlayer located toward a bottom surface of the sensor plate device,undergoes a detectable change so as to indicate the presence ofmicroorganism colonies. Finally, the sensor plate device is inspectedmanually or automatically to determine the presence and location ofmicroorganism colonies.

The instrument performs three main functions on the sensor plate: plateincubation, image acquisition/capture, and image processing. Theinstrument provides a controlled environment for incubating plates,which can include a heater if incubation is to take place at an elevatedtemperature from ambient (though an elevated temperature is notnecessary in all situations). A fluid sample is added to the sensorplate device, after which the sensor plate is placed in the instrumentwhere it is subsequently sensed/observed by an image acquisition/capturedevice (e.g. a camera or scanner) during the incubation period.

Images of the bottom of the sensor plate device can be captured atregular predetermined intervals and subsequently analyzed using one ormore image processing techniques and algorithms to determine whether amicroorganism colony is present on the sensor plate. Theimage-processing algorithm implemented to detect and enumeratemicroorganisms is comprised of one or more of the following steps:

a) Image Masking—to isolate the area of interest from extraneous imagedata;

b) Image Subtraction—to isolate the areas of change between two imagestaken at different time intervals;

c) Image Equalization—to amplify the magnitude of the changes appearingin the subtracted image;

d) Image Blurring—to reduce the effects of single pixel noise in theequalized image (low pass filter);

e) Image Contrast and Brightness Enhancement—to further amplifylocalized differences in the filtered image;

f) Image Thresholding (with several thresholds, if required)—to preparethe image for the colony detection/enumeration algorithm; and/or

g) Colony Detection, Enumeration, and Classification—to determine thepresence of microbial organisms on the plate, to enumerate the number ofcolonies on the plate, and/or perform color analysis to classifycolonies on the plate.

Feasibility studies of a number of different types of sensor platedevices have shown good detection results: E. coli detected inapproximately 6 hours, E. facaelis detected in about 10 hours, and S.aureus detected in about 11 hours. FIG. 13 shows the underside of foursensor plates where the sensor layer has undergone a detectable changein those areas of the sensor layer proximate to microorganism coloniesin an adjacent immobilization matrix layer. Detected colonies in thestudies subsequently yielded microbial dilutions that were immediatelyusable for further testing.

Susceptibility Plate:

Following detection and/or enumeration of microorganisms in a patientsample, it is often desirable to determine to which antibiotics themicroorganism is susceptible. There are now a number of bacterialspecies which increasingly exhibit resistance to one or more classes ofantimicrobial agents, making it that much more important to performsusceptibility testing. Failure of a particular susceptibility test toaccurately predict antimicrobial resistance in a patient's isolate couldsignificantly impact patient care if an antibiotic is used to which themicroorganism is not susceptible.

As part of the present invention, susceptibility plates are provided forperforming microbial antibiotic susceptibility testing. The plates aredisposable and multi-chambered and are inoculated with a microorganism(any suitable organism such as bacteria, fungi, protozoa, algae orviruses) and anti-microbial agent(s) are applied such that themicroorganism is exposed to a variety of concentrations, or a gradientof each anti-microbial agent. The plates are then placed in theinstrument, which monitors and measures the growth (or lack thereof) ofthe microorganisms. This data is used to determine the susceptibility ofthe microorganism to the antibiotics. Such a system automatesantimicrobial susceptibility testing using solid media and Kirby-Bauerstandardized result reporting. Thus, the present invention provides alevel of automation for susceptibility testing previously associatedonly with broth microdilution testing, while retaining the advantages ofthe manual disk diffusion test.

One embodiment of the susceptibility plate of the present invention isillustrated in FIG. 17. Such a plate is provided to be disposable andhave a low cost for manufacture, and is preferably made of plastic. Atop 140 is provided which fits onto bottom 142. Top 140 is preferablytransparent or otherwise having properties that can allow viewing(manually or with machine) of microbial growth in the channels in bottom142. Bottom 142 is provided with a plurality of channels 144 orotherwise mutually isolated chambers. Such channels can be formed withinan insert 146 that fits into bottom 142, or bottom 142 and insert 146can be integrally formed as a single piece (and either or both of thebottom and insert can be opaque). Preferably, marks such as thoseillustrated along each channel in FIGS. 18 and 19 (or other markingssuch as numbers) are provided for aiding in manual measurement of thelength of inhibition in the channel, if such is desired. This is adefinite advantage compared to the standard manual disk diffusionsystem, where calipers are used to measure a diameter of the inhibitionzone, a system that is more labor intensive and less accurate. Eachchannel 144 contains a growth medium that is solid (or semi-solid). Suchgrowth medium may optionally contain an indicator additive for improvingthe readability of growth patterns in the various channels. Also, anindicator may be provided in a separate layer (a “sensor layer”), aconditioning layer may be provided, and various components can beprovided in the gel layer, such as described in above in relation to thesensor plate.

Physically, the external geometry of the susceptibility plate could bemade similar to that of a standard microwell plate (128 mm×86 mm).However, other shapes and sizes are envisioned. The plate could be madeto be almost any geometric shape, including square or even round like astandard agar plate. Whatever the external geometry, internally theplate is partitioned into separate chambers or channels in which thesolid (or semi-solid) medium is held. The wells or chambers within theplate are preferably elongated channels, though triangular, pie-shaped,circular or square wells, or other geometrically shaped wells, are alsoenvisioned. As an example, FIG. 18a illustrates one embodiment wheremutually isolated channels 150 extend almost fully across the width ofthe plate. Antibiotic disks 152 are placed in each channel at the end ormiddle of the channel. Shorter channels 154 could also be formed in theplate, such as those illustrated in FIG. 18b. FIG. 19a illustrates afurther example where antibiotic gradient strips 161 are disposed inelongated channels 163. In this embodiment, an MIC (minimum inhibitoryconcentration) can be determined (the MIC is the concentration at theedge of the inhibition zone—the growth/no growth boundary). Theseparated channels could also be made much narrower, such as illustratedin FIG. 19b. However, if it is desired to use standard antibiotic diskssuch as those commercially provided for standard Kirby-Bauer antibiotictesting (which disks are approximately 6 mm in diameter), then largerantibiotic disk receiving areas should be provided, such as at the endof each narrow channel. Generally, the channel length is greater than 8mm (preferably from 20 mm to 45 mm in length), and the channel width isgreater than 6 mm (preferably from 8 mm to 16 mm in width). A channelwidth of approximately 8 mm is most preferred if standard antibioticdisks are used (which are approximately 6 mm in diameter). Of course, ifantibiotic disks of different size are used, the channel dimensionscould be made larger or smaller. A length of from about 30 to 35 mm ismost preferred as this allows sufficient length to detect and measureinhibition zones resulting from nearly all antibiotic/microorganismcombinations used in susceptibility testing. The depth of the solid orsemi-solid growth medium in the channels should be more than 1 mm,preferably from about 2 mm to about 20 mm, and more preferably fromabout 5 mm to about 15 mm.

One of the purposes of the isolated chambers is to increase the ease andreproducibility of susceptibility testing, as well as to maximize thenumber of tests that can be performed on one susceptibility plate.Whereas standard disk diffusion (Kirby-Bauer) tests are physicallyrestricted to a density of 12 tests or less per 150-mm Mueller-Hintonplate (one test per 14.73-cm²), the present invention easily allows for24 tests or more on a 128 mm×85 mm plate (at least one test per4.53-cm²), a density of more than 3 times that of the standard diskdiffusion plate. Initially it was thought that the length of inhibitionarea in a channel in the present invention would not correlate with theradius of inhibition on a standard disk diffusion plate (when using thesame microorganism and antibiotic). Instead, it was found that themeasured length of inhibition was substantially the same in the presentinvention as the radius measured in the standard plate. By“substantially the same” it is meant that the measured lengths in thepresent invention and in a standard Kirby-Bauer test after the sameperiod of time were either exactly the same, or close enough that theultimate outcome of the test (susceptible, intermediate or resistant asdefined by the National Committee for Clinical Laboratory Standards, orNCCLS) correlated over 80% of the time, and were within the controlranges outlined by the NCCLS. In most cases the results correlated over90% of the time. And, in only in a small percentage (<1.1%) of cases didthe results of the present invention indicate susceptible when thestandard Kirby-Bauer test (with same microorganism and antibiotic)indicated resistant, or vice-versa (<0.9%).

The susceptibility plates of the present invention are used as follows:

1) Disks containing single concentrations, or strips containing severalconcentrations, of each antibiotic being tested are placed (manually orautomatically) onto the inoculated surface of the growth medium in eachchamber of the susceptibility plate. Once the disks or strips are placedon the plate, the antibiotics start to diffuse into the growth mediumforming an antibiotic gradient within the growth medium. Antibioticpanels are flexible and can be user-configurable and/or pre-configured.

2) The susceptibility plate is placed in the instrument (manually orautomatically) where it is incubated, promoting the growth ofmicroorganisms within the chambers, except where inhibited byantibiotics diffused into the growth medium.

3) The susceptibility plate is inspected manually or automatically todetermine the presence and length of inhibition zones in the chambers.Ruler markings or numberings along each channel facilitate manual zonemeasurements. Automatic zone measurements are performed by theinstrument via image capture and image processing.

As mentioned above, the length of the inhibition in the channel of thesusceptibility plate can be measured manually, or automatically. Ifautomatically, an instrument is provided which is responsible forperforming three main functions: susceptibility plate incubation, imageacquisition/capture, and image processing. The instrument provides acontrolled environment to incubate the plates. Susceptibility plates areinoculated and placed in the incubator where they are subsequentlyscanned by an image acquisition device during the incubation period. Theinstrument provides for image acquisition using one or more color and/orgray scale imaging devices: CCD linear array scanner, a CCD line-scancamera, a CCD 2D array camera (still or motion video), a laser scanningcamera, or other device that would provide a sufficiently clear image ofthe susceptibility plate that can be used alone or after furtherprocessing. By “image” it is meant any information, such as opticalinformation, from the susceptibility plate that is ≧a 1×1 pixel. Theimage acquisition is performed at regular preprogrammed intervals, withthe captured image obtained from one or more views and angles of thesusceptibility plate.

The image-processing algorithm implemented to determine susceptibilityis comprised of one or more of the following steps:

h) Image Masking—to isolate the area of interest from extraneous imagedata;

i) Antibiotic Disk or Strip Detection—to determine identity andconcentration of antibiotic under test;

j) Image Subtraction—to isolate the areas of change between two imagestaken at different time intervals;

k) Image Equalization—to amplify the magnitude of the changes appearingin the subtracted image;

l) Image Blurring—to reduce the effects of single pixel noise in theequalized image (low pass filter);

m) Image Contrast and Brightness Enhancement—to further amplifylocalized differences in the filtered image; and/or

n) Inhibition Zone Detection, Measurement, and Inspection—to determinethe susceptibility of microbial organisms to a particular antibiotic.

Results of susceptibility testing with system of the present inventionare shown in FIGS. 20-22. FIG. 20a shows a grayscale image of E. coli ona susceptibility plate (using several different antibiotic disks) taken18 hours after inoculation and antibiotic disk placement. FIG. 20b isthe same plate image after image processing. The image taken atinoculation was subtracted from the image taken after 18 hours ofincubation, the difference image was histogram equalized and blurred,and the zone measurement algorithm was applied to the resulting image.Inhibition zones and equivalent diameter measurements are shown in FIG.20b. Similarly, FIGS. 21a and 21 b show a grayscale image and processedimage for S. aureus using the same timing and processing techniques asfor FIG. 20.

Analyses of images acquired each hour from 1 to 17 hours afterinoculation demonstrate the presence of additional information regardingthe interaction between the microorganisms and the antibiotics.Properties such as the growth rates of the microorganisms, the diffusionrate of the antibiotics, and the characteristics of the antimicrobialeffect on the microorganism are evident. In fact, in a number ofinstances, the inhibition zones are defined as early as 4 to 6 hoursafter plate inoculation and using image processing. As an example, FIGS.22a and 22 b show a grayscale image and processed image, respectively,of Kleb. pneumoniae only four hours after inoculation (FIGS. 22c and 22d are grayscale and processed images, respectively, of the same plateafter 18 hours).

The invention is also envisioned as comprising a top plate and a bottomplate where the bottom plate is a single chamber filled with the solidor semi-solid nutrient medium for the microorganisms and the top plateis provided with a plurality of ribs or dividers. When the top andbottom plates are fitted together (after placement of antibiotic discswithin the “chambers” of the top plate) the nutrient medium is separatedinto isolated chambers as in the embodiment of the invention illustratedin FIG. 4. Of course, the ribs or dividers could be provided as aseparate element from the top plate.

Another aspect of the invention is, rather than determining susceptible,intermediate or resistant, the determination of the MIC. In the presentinvention, MICs can be determined using a regression analysis of thezone measurement (the length of inhibition along the channel), as thediffusion of antibiotic out of the disk forms a highly predictablelogarithmic gradient. Determining the MIC from the length of the diskdiffusion can be performed by any known method, such as that of BIOMIC™System by Giles Scientific (N.Y.).

In accordance with the invention, the antimicrobial agent can be appliedto a plurality of compartments in differing concentrations. The solid orsemi-solid growth medium should be sufficiently solid so that theantimicrobial agent when applied to the growth medium, will diffuse overtime and form a concentration gradient (the concentration gradient canbe formed in a horizontal direction and can continue to diffuse over aperiod of up to 10 to 18 hours or more). The solid or semi-solid growthmedium in each chamber of the susceptibility plate is inoculated (e.g.,swabbed) with a McFarland 0.5 standardized suspension of themicroorganism being tested. If the microorganism is a bacterium, it canbe an aerobic gram-positive organism, aerobic gram negative organism,anaerobic gram positive organism, anaerobic gram negative organism or acell wall deficient organism.

The solid or semi-solid growth medium may comprise one or more ofroutine media, selective media, differential media,selective-differential media, enriched media, susceptibility media,anaerobic media and fungal media. If the media is routine media, it cancomprise one or more of trypticase soy blood agar, trypticase soy agar,tryptic soy, BHI blood agar, BHI agar, Casman blood, HBT bi-layer media,and standard methods agar. If the media is selective media, it cancomprise one or more of, columbia CNA blood, azide blood agar, chocolateselective, Brucella blood, blood SxT, Strep selective I & II, PEA, BileEsculin agar, Clostridium difficile agar, skirrow, CCFA, CLED,Pseudomonas cepacia agar, SxT blood agar, TCBS agar, CIN, Moraxellacatarrhalis media, and charcoal selective. If the media is differentialmedia, it can comprise one or more of brilliant green, CYE—Legionella,centrimide, DNA-se, hektoen enteric agar, Jordans tartrate, mannitolsalt, LIA, TSI, FLO—Pseudomonas F, TECH—Pseudomonas P, Sellers, starchagar, thermonuclease, Tinsdale agar, McCarthy, LSM, sorbitol-McConkey,MUG-McConkey.

If the media is selective and differential media, it can comprise one ormore of MacConkey, EMB, Baird Parker, BHI blood with antibiotics,BiGGY—mycologic, CIN, Clostridium difficile agar, McBride, Pseudomonasisolation agar, S-S agar, tergitol 7, and XLD agar. If the media isenriched media, it can comprise one or more of chocolate, GC chocolate,BHI chocolate, Borget Gengou, heart infusion agar, McCarthy, Regan-Lowe,Thayer-Martin, transgrow medium, cysteine tellurite blood, cysteinetellurite heart, BHT, heart infusion, Loefflers, and serum tellurite. Ifthe media is anaerobic media, it can comprise one or more of columbiabase, PEA, CAN, LKV, BBE, Brucella, BHI blood base, KBE, McClung-Toabe,oxgall, Schaedlers, and Wilkens-Chalgren. And, if the media is a fungalmedia, it can comprise one or more of BHI base, BiGGY, birdseed, cornmeal, cotton seed, DTM, sabourauds dextrose, Fuji medium, inhibitionmold, Littman oxgall, mycologic, mycophil, Nickersons, SABHI, andtrichophytin.

The antimicrobial agents can be one or more of a betalactam antibiotic,a cepheme antibiotic, a glycopeptide antibiotic, an aminoglycosideantibiotic, a macrolide antibiotic, a tetracycline antibiotic, and aquinalone antibiotic. If the antimicrobial agent is a betalactamantibiotic, it can comprise one or more of penicillins,uredopenicillins, synthetics, carbapenems and beta-lactam/inhibitors. Ifit is a cepheme antibiotic, it can comprise one or more ofcephalosporins generations I to IV, and carbacephems. Also, the one ormore antimicrobial agents can comprise one or more of sulfa agents andderivatives, chloramphenicol, clindamycin, nitrofurantoins, polymyxinsand chemical agents.

Identification Plate:

The identification plates as part of the present invention are used foridentifying the microorganism that has been detected in a sample. Theidentification plate is inoculated with a standardized suspension of amicroorganism. The plate is then placed in the instrument. The organisminteracts with various substrates or substrate/indicator combinationsproducing a detectable change in the substrate or indicator. Theinstrument monitors and measures the changes (or lack thereof) of thesubstrates and/or indicators. This data is used to identify themicroorganism.

Physically, the overall dimensions and composition of the identificationplate are envisioned to be similar to that of a conventional 96-wellmicrowell plate: a shallow container that has at least a transparentbottom side. The plate may be flat or have a series of depressions.Specific substrates or substrate/indicator combinations in specific gumsor gels, and placed on the flat surface of the plate bottom, or in theseries of depressions. These gums are capable of absorbing liquid fromthe suspension of organisms. Alternately, the substrates orsubstrate/indicators combinations may be impregnated in an absorbentmaterial such as filter papers discs and affixed to the plate or in thedepressions in the gels or gums. In one embodiment the paper discs areaffixed with xantham gum. The immobilized substrates orsubstrate/indicator combinations may be completely recessed below theoverall plate surface, be level with all overall plate surface, or beraised from the overall plate surface.

The identification plate is used in the following manner.

1. A standardized suspension of microorganisms is prepared from growthplate.

2. The suspension is added to the identification plate.

3. The suspension is localized into multiple sites each containing aspecific substrate or substrate/indicator combination.

4. The identification plate is incubated, promoting the metabolicactivity of the organisms within each site.

5. The change in color or fluorescence in the sites indicates metabolicactivity on the specific substrates.

6. The identification plate is inspected manually or automatically todetermine the pattern of metabolized substrates.

The suspension of microorganisms is applied to one end of theidentification plate. The geometry of the plate is such that thesuspensions flows or is channeled such that all of the localized sitescontaining substrates or substrate/indicator combinations absorb asuffident volume of the suspension. The localized sites where substratesor substrate/indicator combinations are allowed to absorb the suspensionof microorganisms. Excess liquid is removed either by pouring the excessoff the plate or alternately, at the opposite end of the plate fromwhere the suspension was applied, a wicking agent absorbs the excessliquid. In either case, the introduction of sample is a simple one-stepoperation. A lid is applied to prevent evaporation during incubation.

In another embodiment, a three-piece plate is used. This second piecefits above the identification plate and consists of a channel or seriesof channels, each containing “funnels” which allow the suspension toflow to localized sites containing substrates or substrate/indicator,combinations. Again, excess liquid may be poured off or absorbed by awicking agent at the opposite end of the plate from where the suspensionis applied. This channel/funnel piece may either be left in place ordiscarded prior to incubation.

The purpose of sequestering the substrates or substrate/indicatorcombinations is to provide for a localized reaction free frominterference caused by competing substrates. The substrates must be ofsufficient concentration and remain in the localized site during theaddition of the microorganism suspension. The substrates orsubstrate/indicator combinations may be of several types. Specificsubstrates may include o-nitrophenyl-β-D-galactoside and othernitrophenyl derivatives. Alternately, the substrates could include5-bromo-4-chloro-3-indoyl-β-D-galactoside (X-gal) or other “X”derivatives, fluorescent 4-methyllumbelliferyl derivatives or7-amino-methylcoumarin derivatives. Substrate/indicator combinations mayinclude substrates such as sugars (glucose, lactose etc.), amino acids(lysine, arginine, etc.), fatty acids (oleic acid, palmitic acid, etc.),low molecular organic acids (citric acid, glucuronic acid, etc.)poly-alcohols (glycerol, sorbitol, etc.) and others. Indicators mayinclude, but are not limited to pH indicators such as bromcresol purple,phenolphthalein, etc. or oxidation/reduction (red-ox) indicators. Apositive reaction produces a change in either the ultraviolet, visual orinfrared spectrum.

For analysis of the identification plate, images are acquired from thetop and/or bottom sides of the plate at regular intervals (typically,one hour). Scanning at regular intervals provides kinetic substratedata, which may be used to help characterize the microorganisms. Theimage processing algorithm implemented to determine identification iscomprised of the following steps, some of which may be omitted if suchprocessing is not required:

Image Masking (if necessary)—to isolate the area if interest fromextraneous image data

Substrate Zone recognition (if necessary)—to determine location of thesubstrate under test

Image Subtraction (if necessary)—to isolate the areas of change betweentwo images taken at different time intervals

Image Equalization (if necessary)—to amplify the magnitude of thechanges appearing in the subtracted image

Image Blurring (if necessary)—to reduce the effect of single pixel noisein the equalized image (low pass filter)

Image Contrast and Brightness Enhancement (if necessary)—to furtheramplify localized differences in the filtered image

Image Pattern Recognition—to determine which substrates have reacted

An identification of the organism is then accomplished by comparing thereactions of the test organism suspension to known reactivity withdifferent organisms.

FIG. 23a is an image (visible) of an identification plate with aplurality of wells at 24 hours, whereas FIG. 23b is an UV image at 24hours. FIG. 24 is a view of one embodiment of the identification plate310 where a plurality of wells 300 are provided which may have channels305 therebetween. A transparent surface can be provided on the bottom ofeach well, with the remainder of the plate bottom being opaque. A topfor the plate can be provided which allows a method of sealing selectedwells for anaerobic reactions. The container can be provided with animpermeable top to prevent loss of water vapor, and can be designed tobe break-resistant and non-toxic to microorganisms. Barcodes can also beprovided on the identification plate (as well as the other types ofplates) to indicate information such as what type of plate it is, whichpatient, etc.

When a sample is added to the plate 310, the liquid is washed over eachwell 300 via channels 305. Excess liquid can be collected in well 307,in which may be a disposed a sponge or other liquid absorption material.Each substrate/indicator matrix layer is capable of absorbing andretaining 0.05 to 0.1 ml of liquid sample, and preventcross-contamination of substrates during inoculation. Thesubstrate/indicator is preferably non-toxic to microorganisms and has ashelf life of at least one-year.

Urine Screening Plate:

Although second to respiratory infections in rate of occurrence, urinarytract infections account for the most frequently requested testing inthe clinical microbiology laboratory. Clinical urine screening generallyincludes steps such as streaking an established quantity of urine,typically 1 to 10 microliters, onto plates of both a nonselectivemedium, typically blood agar, and a selective and/or differential mediumsuch as MacConkey agar, eosin methylene blue (EMB) agar. Afterincubation, the plates are examined for any microorganism growth,indicating the presence of microorganisms in the sample urine and apossible infection in the patient. Since both the number and type oforganisms present in the specimen are clinically relevant, the readingmust be done by a trained technician, which can be time consuming andexpensive. If required, follow-up testing is done for a more preciseidentification and antibiotic susceptibility profile.

The instrument of the present invention significantly lightens the loadof microbiological testing for bacteriuria. It is capable of detectingand enumerating microorganism colonies on virtually any solid orsemisolid medium, whether the medium is opaque, transparent ortranslucent. The media of choice may include nonselective media such asblood agar and tryptic soy agar, or it could include a selective and/ordifferential media such as MacConkey agar, EMB agar,cystine-lactose-electrolyte deficient (CLED) agar, as well as media withbiochemical substrates added for enhanced differentiation, such asCHROMagar Orientation. Detection and/or enumeration of microbialcolonies can be done either by direct imaging of microbial growth, orimaging of direct or indirect changes that the microorganisms make inthe media, in indicators, or in a sensor layer. Any combination of mediatypes for culture and detection can be placed in separate compartmentsor plates for use in the present invention. Preferably, a two chamberedplate is used which contains one area of nonselective media and one areaof selective and/or differential media. The combination of nonselectiveand differential media allows a measure of both the total number ofmicroorganisms present and some degree of identification, depending onthe specific media selected.

Plates for urine screening are shown in FIGS. 25a and 25 b. For analysisof this type of plate, images are acquired from the top and/or bottom ofthe plate at regular intervals (e.g. every hour). Or, a single image canbe acquired at a predetermined time (e.g. after 24 hours). Though only asingle image is necessary to detect and enumerate colonies on a urinescreening plate, scanning at regular intervals provides dynamic growthdata, which can be used to help characterize the microorganisms. Forexample, in FIGS. 25a and 25 b, K. pneumoniae and E. fecalis developsimilar color but can easily be distinguished by the fact that K.pneumoniae colonies grow at a much greater rate.

The image processing algorithm implemented to detect and enumeratemicrobial growth on the urine screening plate is comprised of thefollowing steps:

a) Image Masking—to isolate the area of interest from extraneous imagedata;

b) Image Subtraction (if required)—to isolate the areas of changebetween two images taken at subsequent time intervals;

c) Image Equalization (if necessary)—to amplify the magnitude of thechanges appearing in the subtracted image;

d) Image Blurring (if required)—to reduce the effects of single pixelnoise in the equalized image (e.g. a low pass filter);

e) Image Contrast and Brightness Enhancement (if required)—to furtheramplify localized differences in the filtered image;

f) Color Separation and Analysis (pre-detection or post-detectionprocessing)—to differentiate the types of microbial colonies(indicator-dependent);

g) Image Thresholding (if required)—to prepare the image for the colonydetection/enumeration algorithm; and

h) Colony Detection and Enumeration—to determine the presence ofmicrobial organisms on the plate, and to enumerate the colonies on theplate.

Studies of the urine screening plates have shown that the colonies canbe detected and enumerated at around 12 to 20 hours after inoculation.FIGS. 25a and 25 b are raw scanned images taken at 18 hours. Themicroorganisms plated were E. faecalis (small, light green—when viewedin color), E. coli (red), S. aureus (small, yellow), P. aeruginosa(medium, yellow), Enterobacter cloacae (large, dark green), andKlebsiella pneumoniae (medium, medium green).

As can be seen by the above description of the present invention, afully-integrated microbial system is provided—essentially one systemthat performs nearly all of the functions required by a clinicalmicrobiology laboratory (microbial detection/screening and/orenumeration, microbial identification, antibiotic susceptibilitytesting, and urine screening). No other fully integrated system is knownto exist. Instead, present systems are actually part of a “family” ofnon-integrated instruments/products where multiple instruments must bepurchased (and laboratory space must be found for each) to accomplishthe multiple functions of the present invention. The present inventionis easier to use and maintain than several separate instruments, it isless expensive to own and operate a single instrument, the presentinvention requires less laboratory space than several separateinstruments, and it is modular and flexible to accommodate a widecapacity range. Not only can the present invention perform multiplefunctions within the same instrument, but also it can perform thesemultiple functions at the same time (different types of plates can bedisposed adjacent each other). This allows the laboratory user a widedegree of flexibility in scheduling needed tests.

The foregoing description is sufficient to enable one skilled in the artto practice the invention. The examples herein should not be construedas limiting the scope of the claims in any way. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

We claim:
 1. A method for performing a plurality of microbiologicaltests in any order or at the same time, and within the same instrument,comprising: a) performing microbiological detection and/or enumerationtesting, wherein one or more test samples are added to respectivedetection plates, each of said plates is placed on or within a holder ofthe instrument and incubated in the instrument, and during and/or afterincubation, each plate is imaged by an imaging device in the instrumentin order to determine whether and/or how many microorganism colonies arepresent; b) performing susceptibility and/or identification testing ofmicroorganisms, wherein if susceptibility testing is performed, one ormore samples containing microorganisms are added to respectivesusceptibility plates before or after one or more antibiotics are addedto the plate, each of said plates is placed on or within a holder of theinstrument and incubated in the instrument, and each plate is imaged byan imaging device the same as or different as the imaging device of step(a) to determine whether and/or the extent of microorganism growthinhibition, and if identification testing is performed, one or moresamples containing microorganisms are added to respective identificationplates containing substrates for microorganisms, each of said plates isplaced on or within a holder of the instrument and incubated in theinstrument, and each plate is imaged by an imaging device the same as ordifferent as the imaging devices in steps (a) and/or (b) to classifyand/or identify the microorganism(s) on each plate.
 2. The methodaccording to claim 1, wherein said imaging device for imaging said oneor more detection plates is the same imaging device for imaging said oneor more susceptibility and/or identification plates.
 3. The methodaccording to claim 1, wherein each of said detection plates and each ofsaid susceptibility and/or identification plates are of the samedimensions and are constructed so as to be interchangeably held by saidholders of said instrument.
 4. The method according to claim 1, whereineach plate has on a surface thereof, information for identifying thesource of the sample thereon and/or the one or more tests to be run onsaid sample.
 5. The method according to claim 4, wherein saidinformation is in the form of a barcode which can be read either by theimaging device or a barcode reader.
 6. The method according to claim 1,further comprising one or more transport mechanisms for moving saidplates and said one or more imaging devices relative to each other. 7.The method according to claim 6, wherein the same imaging device ordevices and the same transport mechanism or mechanisms are usedregardless of the type of plate being imaged or moved.
 8. The methodaccording to claim 1, wherein said incubation of said detection platesis performed in the same incubator as for the incubation of thesusceptibility and/or identification plates.
 9. The method according toclaim 6, wherein said plates are moved relative to said imaging devices.10. The method according to claim 6, wherein said one or more imagingdevices are moved relative to said plates.
 11. The method according toclaim 6, wherein both said plates and said one or more imaging devicesare moved relative to each other.
 12. The method according to claim 1,further comprising monitoring and controlling the instrument functionsvia a user interface.
 13. The method according to claim 1, wherein saidimaging of said plates is performed at regular intervals.
 14. The methodaccording to claim 13, wherein the imaging of said plates is performedby one or more color and/or gray scale imaging and illumination devices.15. The method according to claim 1, wherein the imaging of said platesis performed by a plurality of imaging devices.
 16. The method accordingto claim 1, wherein said plates are moved stepwise to imaging stationswhere they are imaged by said imaging devices.
 17. The method accordingto claim 1, wherein said imaging is performed by one or more of a CCDlinear array scanner, a CCD line-scan camera, a CCD 2D array camera, anda laser scanning camera.
 18. The method according to claim 17, whereinsaid imaging comprises capturing an image of each plate at one or moreviews or angles of each plate.
 19. The method according to claim 1,wherein after an image is captured of a plate, the image is processed byusing one or more of the following image processing steps: colorseparation and selection, image masking, image subtraction, imageequalization, image blurring, image contrast and brightness enhancement,and image thresholding.
 20. The method according to claim 19, furthercomprising: a) colony detection, enumeration and/or classification, ifthe plate is a detection or screening plate; b) inhibition zonedetection and measurement, if the plate, or a part of the plate, is forantibiotic susceptibility; and/or c) substrate reaction zone inspection,if the plate, or part of the plate, is for microbial identification. 21.The method according to claim 1, wherein said microbiological detectionand/or enumeration testing, said susceptibility testing, and saididentification testing are performed sequentially and/or concurrently inthe instrument.
 22. The method according to claim 1, wherein saidsusceptibility testing and said identification testing are performed onthe same plate for the same microorganism being tested.
 23. The methodaccording to claim 1, wherein said microbiological detection and/orenumeration testing is performed on one or more detection plates, eachhaving a sensor layer adjacent a microorganism immobilization layer,wherein said imaging of each detection plate is performed on a side ofeach detection plate proximate to said sensor layer, said sensor layerundergoing a detectable change if microorganisms are present in saidimmobilization layer, and the imaging device capable of detecting thechange in the sensor layer of the detection plate.
 24. The methodaccording to claim 1, wherein said susceptibility testing is performedon a plate having at least a portion thereof devoted to determining thesusceptibility of a microorganism thereon, said plate having a pluralityof channels with antibiotics in each channel and a test sample added toeach channel, wherein said imaging is performed on said plate so as todetermine the existence and/or degree of inhibition of microorganismgrowth in each channel.
 25. An instrument for performing a plurality ofmicrobiological tests in any order or at the same time, comprising: a)one or more plate holders for holding detection plates, and for holdingsusceptibility and/or identification plates; b) a plurality of detectionplates for performing microbiological detection and/or enumerationtesting, the detection plates held on one or more of said plate holders;c) a plurality of plates for performing susceptibility and/oridentification testing of microorganisms, said susceptibility and/oridentification plates held on one or more of said plate holders; d) oneor more imaging devices provided for imaging plates at one or more timeperiods; e) one or more transport mechanisms for moving said one or moreplate holders and/or one or more imaging devices relative to each other;f) a computer processor for controlling instrument functions and forprocessing images obtained by said imaging devices so as to detect thepresence of microorganisms on said detection plates and so as todetermine antibiotic susceptibility and/or microbial identification onsaid plates for antibiotic susceptibility and/or microbialidentification.
 26. The instrument according to claim 25, wherein saidone or more plate holders are elongated trays constructed so as to holda plurality of plates thereon.
 27. The instrument according to claim 25,wherein said plate holders are constructed so as to be capable ofholding detection plates and susceptibility and/or identification platesat the same time.
 28. The instrument according to claim 27, wherein saidplate holders are constructed so as to be capable of holding detectionplates interchangeably with antibiotic susceptibility and/oridentification plates.
 29. The instrument according to claim 28, whereinsaid plate holders are constructed so as to each hold detection plates,antibiotic susceptibility plates, and identification plates at the sametime.
 30. The instrument according to claim 25, wherein said detectionplates are plates each having therein a solid or semi-solidimmobilization layer.
 31. The instrument according to claim 30, whereinsaid detection plates further comprise a sensor layer which is capableof undergoing a detectable change due to the presence of microorganismsin the immobilization layer.
 32. The instrument according to claim 31,wherein said sensor layer is opaque.
 33. The instrument according toclaim 25, wherein said testing for antibiotic susceptibility and saidtesting for microbial identification are performed on the same orseparate plates.
 34. The instrument according to claim 33, wherein forantibiotic susceptibility, at least a portion of a plate is comprised ofplurality of channels or wells having therein a solid or semi-solidnutrient medium.
 35. The instrument according to claim 34, wherein eachchannel or well further comprises an antimicrobial agent therein. 36.The instrument according to claim 33, wherein for microbialidentification testing, at least a portion of a plate is comprised of aplurality of channels or wells having therein different microbialsubstrates.
 37. The instrument according to claim 25, further comprisingurine screening plates for performing urine screening.
 38. Theinstrument according to claim 25, wherein said one or more imagingdevices are devices capable of collecting information from each platethat is greater than or equal to a 1×1 pixel.
 39. The instrumentaccording to claim 38, wherein said one or more imaging devices arecomprised of at least one of a CCD linear array scanner, a CCD line-scancamera, a CCD 2D array camera, or a laser scanning camera.
 40. Theinstrument according to claim 38, wherein at least two imaging devicesare provided, one disposed for imaging a top side of each plate, and asecond for imaging a bottom side of each plate.
 41. The instrumentaccording to claim 25, wherein said one or more imaging devices comprisea light source for illuminating each plate.
 42. The instrument accordingto claim 25, further comprising an incubator for maintaining anenvironment conducive to microbial growth.
 43. The instrument accordingto claim 25, wherein said processor performs one or more of thefollowing image processing steps: color separation and selection, imagemasking, image subtraction, image equalization, image blurring, imagecontrast and brightness enhancement, and image thresholding.
 44. Theinstrument according to claim 43, wherein said processor performs: d)colony detection, enumeration and/or classification, if the plate is adetection or screening plate; e) inhibition zone detection andmeasurement, if the plate, or a part of the plate, is for antibioticsusceptibility; and/or f) substrate reaction zone inspection, if theplate, or part of the plate, is for microbial identification.
 45. Amethod for detecting and/or enumerating microorganism colonies,comprising: a) inoculating one or more detection plates, each detectionplate comprising an immobilization layer and a sensor layer whichundergoes a detectable change when microorganisms are present in theimmobilization layer; b) placing said one or more microbial detectionplates on one or more respective plate holders c) incubating saiddetection plates for a period of time sufficient to allow formicroorganism growth and a corresponding change in the sensor layer ofeach plate having microorganisms therein; d) taking an image of thesensor layer of each detection plate with an imaging device; and e)processing each sensor layer image to determine whether and/or how manymicroorganism colonies are present on each plate.
 46. A method forperforming antibiotic susceptibility testing comprising: a) adding atest sample to one or more channels of one or more antibioticsusceptibility plates, each channel comprising a solid or semi-solidnutrient medium therein; b) placing said one or more susceptibilityplates onto one or more plate holders; c) incubating said one or moresusceptibility plates; d) taking an image of each channel of eachsusceptibility plate; and e) processing each image so as to determinethe existence of or extent of microorganism growth inhibition in eachchannel of each plate.
 47. The method according to claim 1, wherein whensaid plates are imaged, light is illuminated onto one or more sides ofeach plate being imaged.
 48. The method according to claim 47, whereinsaid light is broadband and/or narrowband visible, ultraviolet and/orinfrared illumination.
 49. The instrument according to claim 25, furthercomprising one or more light sources for illuminating each plate beingimaged.
 50. The instrument according to claim 49, wherein said one ormore light sources are a source of broadband and/or narrowband visible,ultraviolet and/or infrared illumination.
 51. The instrument accordingto claim 50, wherein said one or more imaging devices comprises said oneor more light sources.